Purification and characterization of extracellular vesicles from tachyzoites of Toxoplasma gondii

Abstract

Toxoplasma gondii, a protozoan that causes toxoplasmosis, is transmitted from animals to humans
through ingestion of infected meat or oocysts released by felines into the environment. In humans, the
infection is usually asymptomatic, but in cases where primary infection occurs during pregnancy it
can lead to neonatal malformations. In patients such as AIDS, reactivation of latent infection occurs,
with episodes of parasite proliferation, causing symptomatic disease, such as cerebral or disseminated
toxoplasmosis. In the last decades, small structures secreted by prokaryotic and eukaryotic cells called
extracellular vesicles (EVs) were studied. These small structures can be isolated by ultracentrifugation,
chromatography and examined by electron microscopy. EVs participate in the communication between
cells, transfer of proteins, lipids and nucleic acids. In addition, them can carry disease biomarkers,
bioreactive macromolecules contributing to the pathogenesis of diseases. In view of the above, the
present study aimed to establish a protocol to isolate and characterize extracellular vesicles produced
and excreted by tachyzoites of T. gondii RH strain. To achieve this goal, tachyzoites from VERO cell
cultures were isolated from the culture supernatants and centrifuged in five sets of washes. Next, they
were incubated in culture medium for 24 hours for secretion of the EVs. The size and concentration of the
isolated EVs by particle scan analysis (NTA) were investigated on the NanoSight (NTA) equipment. In
parallel, the morphology and the release of the EVs were also investigated by transmission and scanning
electron microscopy. Then, EVs were purified by gel-exclusion chromatography in 1 ml aliquots (24-
32 fractions) and immuno-selected by ELISA using a pool of reagent sera for toxoplasmosis. Next, the
presence of miRNA in the EVs was investigated; and finally, the protein profile of T. gondii EVs was
evaluated and verify by serological tests if these particles could be recognized by the host immune
system. The results showed that the protocol for recovery of T. gondii EVs was established from 1 to
1010 tachyzoites obtained from cultured cells. Analyzes performed by NTA allowed us to determine
that about 1 x 106 tachyzoites secreted 4 to 8 x 108 EVs / mL within 24 hours of incubation in culture
medium. Additionally, these vesicles presented morphology and size of 165-175 nm of diameter,
corresponding microvesicles size. These results were also confirmed by the evaluation of images
provided by transmission and scanning electron microscopy. The miRNA purifications from the EVs
and subsequent microfluidic electrophoretic run confirmed the presence of smallRNA and miRNA in
the vesicles. SDS-PAGE analyzes show that the proteins carried by the EVs showed an electrophoretic
profile with a spectrum of 15 to 70 kDa. Sera from chronically infected mice (with 2 different strains of
T. gondii) and humans recognized distinct electrophoretic patterns in immunoblotting.